Defined sequence RNA synthesis in the 3′→5′ direction is now well established and currently in use for synthesis and development of a vast variety of therapeutic grade RNA aptamers, tRNAs, siRNA and biologically active RNA molecules. The chemical synthesis of RNA is desirable because it avoids the inefficiencies and limitation of scale of synthesis such as by in vitro transcription by T7 RNA polymerase (See Helm, M., et al., RNA, 5:618-621, 1999, the entire teachings of which are incorporated herein by reference). Chemical synthesis of RNA is desirable for studies of RNA structure and function, and many useful modifications can be achieved selectively, such as site specific introduction of functional groups; viz., disulphide cross linking as a probe of RNA tertiary structures (See Maglott, E. J., Glick, G. D., Nucl. Acids Res., 26: 1301-1308, 1999, the entire teachings of which are incorporated herein by reference).
This approach utilizes a ribonucleoside with suitable N-protecting group: generally 5′-protecting group, the most popular being dimethoxytriphenyl, i.e., the DMT group; 2′-protecting group, out of which most popular is t-Butyldimethylsilyl ether; and, a 3′-phosphoramidite, the most popular of which is cyanoethyl diisopropyl. This component is then coupled with a nucleoside with a suitable N-protecting group, 2′ or 3′ succinate of a ribonucleoside attached to a solid support. The coupling of component 1 and 5′-OH-n-protected-2′,3′-protected-nucleoside are also achieved in solution phase in presence of an activator leading to dimers and oligoribonucleotides, followed by oxidation (3′→5′ direction synthesis), also leads to a protected dinucleotide having a 3′→5′-internucleotide linkage, Ogilvie, K. K., Can. J. Chem., 58: 2686, 1980 (scheme 1).
A number of such synthetic RNA require a modification or labeling of the 3′-end of an oligonucleotide. The synthesis of 3′-end modified RNA requiring lipophilic, long chain ligands or chromophores, using 3′→5′ synthesis methodology are challenging, difficult to synthesize and generally result in low coupling efficiency and lower purity of the final oligonucleotide, in general. Additional purifications are generally required. Therefore, new synthetic methodologies are needed to synthesize RNA molecules quickly, and cleanly and in a form that allows for modification at the 3′ end.